Host immune response

Adenoviruses elicit a strong immune response along with increased transduction and replication inside the tumors. This can be a disadvantage for adenoviral therapy, leading to rapid clearance of the virus and preventing virus replication and spreading inside the tumor (Prestwich et al., 2009). New strategies for engineering adenoviral vectors have been employed to overcome these immunological barriers (Cerullo et al., 2010; Koski et al., 2010; Loskog et al., 2005). Host defense mechanisms towards adenoviruses can be classified in innate and adaptive immune responses. For the adaptive immunity, the host has four options to respond to Ad: 1) cellular immune responses mediated by T cells; 2) humoral response orchestrated by B-cells and leading to the production of neutralizing antibodies; 3) production of interferons (IFNs) to ablate the intracellular activities of the invading virus; and 4) induction of apoptosis by switching into proapoptotic proteins.

1.6.2.1 Innate immune responses

The innate immune response is the host’s first line of defense. Ads induce the innate responses immediately after infection (Raper et al., 2003; Zhang et al., 2001). The induction of the innate immune response following adenovirus infection has been well studied both in vitro and in vivo (Cerullo et al., 2007; Muruve et al., 2004; Tuve et al., 2009). It became of great interest, in particular because of the one and only lethal adverse effect reported with Ad and thought to be due to innate immune response, which provoked cytokine storm, intravascular coagulopathy and multiorgan failure (Raper et al., 2003). Even though many improvements have been made to understand the mechanisms of interaction of viruses with the innate immune system, still little is known. The innate immune response is the major player for the clearance of adenovirus from the body (Lenaerts et al., 2008). Host cells have a range of strategies to overcome any danger signal by releasing specific cytokines and chemokines, leading to recruitment of neutrophils responsible for the inflammatory response (Muruve et al., 1999).

Neutrophils recruited at the site of infection produce cytokines which lead to amplification of the antiviral immune cascade. At the same time, recruitment of macrophages and natural killer (NK) cells and activation of complement are important factors for the clearing of the adenovirus (Worgall et al., 1997). Macrophages, and specially monocytes, are phagocytes which release antiviral cytokines and effectively

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present antigens necessary for induction of adaptive immune response (Guidotti and Chisari, 2001). On the other hand, NK cells spontaneously kill MHC-I deficient tumor cells (Whiteside and Herberman, 1995). They mediate the cytotoxicity via perforin and induction of different cytokines. As described by Smyth and colleagues, NK cells are highly responsive to many cytokines such as IL-2, IL-12, IL-15 and IFNs, and they increase their cytolytic, secretory, proliferative and anti tumor activities (Smyth et al., 2001). More recently, an increasing body of evidence is pointing out the importance of the Toll-like receptor family (TLRs) as a major factor in modulating the innate immune response towards adenoviruses (Cerullo et al., 2007). TLRs interact with various viral components triggering part of the immune response to adenoviral vectors. TLR9, an endosomal receptor, is activated by double strand DNA (dsDNA). This receptor is able to sense viral infection at cellular levels and triggers cytokines expression as a response (Cerullo et al., 2007). In addition, TLR2, expressed on cell membrane, is also able to sense a viral infection eliciting part of the characteristic immune response to the adenovirus (Suzuki et al., 2010). Another function of the innate immune system is recognition of structures or products known as pathogen-associated molecular patterns (PAMPs) through a set of receptors called pattern-recognition receptors (PRRs) (Akira et al., 2006). The best studied receptors from this family are TLRs and NOD-LRR (nucleotide binding oligomerization domain/leucine-rich repeat) (Huang and Yang, 2009). These ubiquitous receptors are particularly abundant on dendritic cells and macrophages. This recognition triggers a series of events that finally eradicate viral infection. A principal mechanism for this is mediated through Nf-κB activation which signals via mitogen activated protein kinase (MAPK) pathway and results in transcription of different chemokines and cytokines of the host cell (Ferreira et al., 1999; Girardin et al., 2002; Inohara and Nunez, 2003). Finally, the activation of complement is also an important innate defense mechanism of the host to enhance viral clearance. Appledorn and colleagues demonstrated that complement C3 knock-out mice have a reduced cytokine production upon stimulation with adenovirus (Appledorn et al., 2008).

1.6.2.2 Adaptive immune responses

During the last decade, scientists have discovered new receptors of the innate immune system that can shape the adaptive immune response. Still, there is no such

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distinct line between innate and adaptive immunity. These two processes are cross-linked and cannot exist separately. Adaptive immunity is a complex process orchestrating different mechanisms including: cellular immune responses, humoral responses, the role of IFNs in bridging the innate with adaptive response and induction of apoptosis mediated by effector cells.

1.6.2.2.1 Cellular immune response

Cellular immune response against tumors is orchestrated by T cells, and is a balance between induction of anti-tumor response and clearance of virus itself. Several studies demonstrated that cellular immune response towards virus elimination is mainly T cell mediated along with induction of the humoral response and production of IFNs (Russell, 2000; Schagen et al., 2004). T cell mediated response involves both cytotoxic CD8+ and helper CD4+ cells. After the uptake of adenovirus, viral proteins and transgenes are expressed, processed into small oligopeptides and presented on the cell surface. These antigens are recognized by CTLs in a complex with class I proteins of the MHC on the surface of the cell. The binding of CD8+ T cells to this peptide-MHC complex I leads to formation of specific CTLs towards Ad or transgene product (LacZ for instance) (Schagen et al., 2004). Further, the cellular immune response is engaged by CD4+ T helper (Th) cells primarily belonging to Th1 subset (Yang and Wilson, 1995; Yang et al., 1995). These CD4+ cells, in contrast of CD8+ cells, are activated by antigens from the input virions.

These antigens are presented through the MHC class II molecules on the surface of the antigen presenting cells. Activated CD4+ cells start to produce IL-2 and IFN- γ (Maraskovsky et al., 1989). These cytokines belonging to Th1 subset, further on induce CD8+ cells differentiation into cytotoxic CD8+ cells (CTLs) (Wille et al., 1989). It has been also suggested that activated CD4+ cells can destroy Ad-transduced cells themselves (Yang and Wilson, 1995).

1.6.2.2.2 Humoral response

Besides cellular immune response, another immune adaptive mechanism towards adenovirus is the humoral response. The humoral response is represented by the production of antibodies targeted towards any pathogen incorporated by cells. In case of adenoviral infection, these antibodies are mainly targeted towards adenoviral capsid proteins (Gahery-Segard et al., 1998; Willcox and Mautner, 1976). These antibodies do

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not contribute to virus elimination (Yang et al., 1996) but they prevent adenovirus binding to cells and promote opsonization by macrophages (Schagen et al., 2004). Pre-existing immunity towards wild-type adenovirus occurs in most of the patients. Given this, the humoral immune responses are of importance for planning the dose, route of administration and target tissue.

Humoral response depends on B cell capacity of recognizing viral antigens and producing immunoglobulins. This recognition process is mediated through CD4+ helper cells (Yang et al., 1996). They release immunoglobulins into plasma which specifically recognize the antigens. This process starts with the binding of adenovirus particles to the surface of immunoglobulin of B cells (Schagen et al., 2004). After internalization and processing of the virus, the antigens are exposed on the surface of B-cells through MHC class II molecules (Paul and Seder, 1994). The complex formed can be recognized by the activated T helper cells of the Th2 subset. These activated CD4+ cells start to produce cytokines like IL-4, IL-5, IL-6 and IL-10 which induce B cell transformation into plasma cells (Paul and Seder, 1994). Further on, the plasma cells secrete antibodies which are against adenovirus capsid. Even though it was mentioned that Th1 subset can also induce a small humoral response, this is more involved in antibody-isotype switching (Boom et al., 1988). In conclusion, Th2 cells control the production of Ab isotypes IgG1, IgG2b, IgA and IgE mediated by cytokines like IL-4 while Th1 cells control the switch to IgG2a or Ig3 as a response to IFN-γ secretion (Finkelman et al., 1990; Germann et al., 1995; Schagen et al., 2004).

1.6.2.2.3 Interferons

Interferons are divided in two classes: type I with IFN-α and IFN-β and type 2 with IFN-γ. Interferons are thought to be the bridge between innate and adaptive response to adenoviral infection. They are released very early after virus infection and present certain cell specificity. Type I interferons are thought to play a critical role in both innate and adaptive responses, while type II, IFN-γ, mostly acts for adaptive immune response (Goodbourn et al., 2000). IFN-γ is crucial for many events that occur in tumors, including up-regulation of pathogen recognition, antigen processing and presentation, regulation of the antiviral state, inhibition of cellular proliferation and induction of apoptosis, immunomodulation, and leukocyte trafficking (Schroder et al., 2004). The mechanism of

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action for IFNs is mediated through Jak/STAT pathway (Look et al., 1998). Interferons bind to cellular receptors which leads to formation of STAT complexes (Paulson et al., 1999). These complexes are transferred to the nucleus where they bind to interferon-response elements of the cellular DNA and inhibit the intracellular activities of the invading virus (Randall and Goodbourn, 2008).

1.6.2.2.4 Apoptosis

Another strategy for the human body to overcome the viral infection is induction of apoptosis in infected cells. A major player in this process is p53 tumor suppressor protein. This protein regulates the transcription of specific genes which are involved in cell cycle arrest and apoptosis. However, adenoviral gene E1B-19k gene can counteract the proapoptotic effect of the p53 (Han et al., 1996).

Another mechanism for inducing apoptosis is complemented by TNF-α production (Elkon et al., 1997). This cytokine is immediately secreted by macrophages and leukocytes as a response to viral infection. TNF-α plays an important role in virus clearance from the body through direct induction of caspase pathway (Russell, 2000).

Additionally, Fas and Fas-ligand are also involved in the induction of apoptosis. These proteins are reported to be the major mediators in adenovirus elimination from the liver (Chirmule et al., 1999). Adenoviral proteins encoded in the E3 region cause Fas to be removed from the cell surface and degraded. FIP-3 protein gets activated and blocks the NF-κB release, and the proapoptotic pathway is inhibited (Li et al., 1999). Moreover, E1A can induce direct apoptosis mediated through caspase-8 pathway, independent of p53 presence (Putzer et al., 2000).